Expression of human placental-type 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase in various cells and cell lines.
The expression of the human placental-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) in various human cells and cell lines was investigated at the levels of transcription and translation. Analyses by both Northern blotting and a reverse transcription-polymerase chain reaction (RT-PCR) showed that BeWo, U-937, SupT1, H9, HeLa, HepG2, and human mononuclear cells, as well as human placental chorionic cells, expressed HP2K mRNA. All the nucleotide sequences of RT-PCR products from these cell lines were identical to that of HP2K. The expression of HP2K protein was determined by Western blot analysis of fractions from POROS-HQ column chromatography of the cell extracts from U-937 cells, which was used as an example of HP2K-mRNA positive cell lines. As with the 6-phosphofructo-2-kinase activity of HP2K, the activity of 6-phosphofructo-2-kinase in extracts of U-937 cells was not inhibited by glycerol 3-phosphate, a known 6-phosphofructo-2-kinase inhibitor of liver- and testis-type isozymes. These results strongly suggested that various cell lines, in particular U-937 cells, express functional HP2K enzyme. Furthermore, 6-phosphofructo-2-kinase in U-937 cells was found to be activated by treatments with isoproterenol and phorbol 12-myristate 13-acetate, indicating regulation of 6-phosphofructo-2-kinase activity in U-937 cells by protein kinases A and C.
Hirata
T
T
Department of Biochemistry, School of Pharmaceutical Sciences, Nagasaki University, Japan.
Kato
M
M
Okamura
N
N
Fukasawa
M
M
Sakakibara
R
R
D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D006597:Fructose-Bisphosphatase; D005786:Gene Expression Regulation / Q000235:genetics*; D005994:Glycerophosphates / Q000494:pharmacology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D010732:Phosphofructokinase-1; D025481:Phosphofructokinase-2; D010920:Placenta / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000235:genetics; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics
D005994:Glycerophosphates; D012333:RNA, Messenger; C029620:alpha-glycerophosphoric acid; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D010732:Phosphofructokinase-1; D006597:Fructose-Bisphosphatase; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate